Multiple accessory-genes sequence typing (MAST): pan-genome-guided PCR target discovery enables species-level...

Salmonella is a major zoonotic bacterial pathogen with significant impacts on public health, food safety, and veterinary medicine. The genus Salmonella includes exactly two recognized species, S. enterica and S. bongori. Most routine molecular surveillance assays target conserved genus-level genes, such as invA, which do not distinguish Salmonella enterica and Salmonella bongori, potentially obscuring species-specific ecology and source attribution. Here, we present the first introduction of multiple accessory-genes sequence typing (MAST), a pan-genome-guided framework for binary (two species) bacterial PCR target screening, primer design, and experimental validation, demonstrated here in Salmonella. Applying MAST to 2236 high-quality Salmonella genomes, we identified sopD2 as a species-discriminative locus. sopD2-targeted primers with the highest MAST Diff score (= 0.999) demonstrated high analytical specificity for Salmonella, with no cross-amplification observed against Escherichia coli, Streptococcus suis, or Pasteurella multocida, and enabled clear PCR differentiation of S. enterica from S. bongori across laboratory isolates. Taken together, our results position MAST as a broadly applicable and practical pipeline for species-level PCR marker discovery across diverse bacterial taxa and validate sopD2 as a robust target for discriminating Salmonella species, enhancing the accuracy of molecular surveillance and the clarity of source attribution.